Chromatin analysis

Blocking solution for Protein-G and -A Sepharose Beads Fast Flow 4 (GE Healthcare): 50µl of beads (25µl ProteinG plus 25µl ProteinA) per sample, blocked in 15ml 5% BSA in PBS at 4°C for at least 1h.

BSA (fraction V, Roth): 5% BSA in PBS; the solution was aliquoted in 50ml falcon tubes and stored frozen at -20°C.

Formaldehyde: 37% stock (Merck); 1% Formaldehyde in PBS working solution: 676µl formaldehyde in 25ml PBS.

Glycine (Merck); 0.125M glycine in PBS (235mg glycine in 25ml 1XPBS). PBS: 137mM NaCl; 2.7mM KCl; 8mM Na2HPO4; 1.7 mM KH2PO4; pH 7.2.

RIPA buffer: 50mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% Na-Deoxycholate, 150mM NaCl, 1mM EDTA, 0.1% SDS, 0.5mM DTT (to be add freshly), 5mM Na-Butyrate (to be add freshly), Protease inhibitor cocktail (Roche; 1 tablet per 100ml RIPA solution); the solution was kept at 4°C.

TES buffer: 50mM Tris-HCl pH 8.0, 10mM EDTA, 1% SDS, 50mM NaHCO3 (to be added freshly); the solution was kept at RT.

Wash buffer I: 20mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2mM EDTA, 150mM NaCl.

Wash buffer II: 20mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2mM EDTA, 500mM NaCl.

Wash buffer III: 10mM Tris-HCl pH 8.0, 0.25M LiCl, 1% NP-40, 1% Na- Deoxycholate, 1mM EDTA.

Wash buffer IV: 10mM Tris HCl pH 8.0, 1mM EDTA.

Proteinase K/Glycogen solution: 10mg/ml Proteinase K, 20mg/ml Glycogen (Fermentas); mix 10 parts of proteinase K with 2 parts of glycogen.

3.9.2 Chromatin Immunoprecipitation (ChIP)

The ChIP protocol was established for Xenopus based on a published protocol, with the following modifications (Blythe, Reid et al. 2009). Aliquots of 50

Xenopus tropicalis embryos per condition (wildtype or injected) were fixed at NF 14- 15 in 5ml 1% formaldehyde in PBS for 5min at 20°C on a rolling wheel. Crosslinking was stopped by a 10min wash with 0.125M glycine/PBS, followed by three washes in PBS. Fixed embryos were transferred in 1.5ml eppendorf tubes, frozen in liquid nitrogen and stocked at -80°C. At experimental day1, embryos were cautiously thawned over 15min on ice.

Two 15ml conical tubes of blocked protein-G and -A beads were prepared by incubating the proper amount of beads with 15ml of 5% BSA in PBS. The tubes were incubated at 4 °C while mixing for at least 1h.

600µl of 4°C RIPA buffer was added to each 50 embryos aliquot. Samples were homogenized with a pellet pestle by gently disrupting the embryos until no large embryo fragments are visible. Embryos were incubated on ice at least 10min and subsequently centrifuged at 14,000rpm for 10min at 4°C. The supernatant was discarded and the wall of the tubes was carefully wiped with a kimwipe to remove any lipid contaminant. 650µl of 4°C RIPA buffer was added to each sample; the pellet was re-homogenized vigorously. Samples were subsequently sonicated using the Bioruptor (Diagenode) for 25 cycles, each composed of a 30sec pulse and 30sec rest. Samples were centrifuged at 14,000rpm for 10min at 4°C. 600µl sheared chromatin from two samples were pooled together (in order to obtain 1.2ml sheared chromatin from 100 fixed embryos) and transferred into a pre-chilled, clean 1.5 microcentrifuge tube. Input samples were prepared as followed: in a clean 1.5ml microcentrifuge 195µl TES buffer were combined with 5µl sheared chromatin. Input samples were snap-frozen in liquid nitrogen and stored at -80°C and processed together with the IP-samples, once they were completed.

One of the two 15ml conical tubes containing the blocked protein-G and -A beads was centrifuged at 1000rpm for 5min at 4°C. Excess of 5% BSA/PBS was removed and the beads were gently resuspended by pipetting. A pre-clearing step was achieved by dispensing 50µl blocked beads to each sample of sheared chromatin and incubating each sample at 4°C with mixing for 1-1.5h. Samples were subsequently centrifuged at 1000rpm for 1min at 4°C. Each 1.2ml sheared chromatin sample was separated into two samples by transferring 580µl of pre-cleared,

sheared chromatin in two new 1.5ml pre-chilled, clean microcentrifuge tubes. Each new tube was filled with RIPA buffer and the immunoprecipitation was begun by adding 5µg of antibody to only one of the two tubes, keeping the second one as negative control. Samples were incubated overnight at 4°C with mixing.

At experimental day 2 the second 15ml conical tube containing the blocked protein-G and –A beads was centrifuged at 1000rpm for 5min at 4°C. Excess 5% BSA/PBS was removed, and the beads were gently resuspended by pipetting. 50µl blocked beads was added to each sample. Samples were incubated at 4°C with mixing for 1.5 hour and afterwards centrifuged at 100rpm for 1min at 4°C. Beads were subsequently washed: each wash consisted of a 1-minute centrifugation at 1000rpm at 4°C to pellet the immunocomplexes; removal of supernatant with a 20- gauge needle; addition of 1ml wash buffer, and final incubation at 4°C on a rotating wheel for 5min. Samples were washed 8 times in total, using 2 washes each with buffers I through IV. Following the washes, the supernatant was aspirated with a 26- gauge needle inserted into the beads to completely removed any residual wash buffer. 200µl TES buffer was added to the beads. Elution was achieved by incubating the samples at 65°C for 1h in a table shaker (1000rpm). During this time the frozen input samples were thawned and vortexed to resuspend any precipitated SDS. All the different samples (input, IP and negative control) were processed in the same manner for the rest of the procedure.

Samples eluted from the beads were centrifuged at 14,000rpm at RT for 1min. 200µl of the eluted supernatant was transferred to a new 1.5ml microcentrifuge tube. RNase treatment was achieved by adding 2µl of 10mg/ml stock RNase A (Quiagen) to each sample and incubation for 45min at 37°C. Subsequently, 12µl of Proteinase K/Glycogen solution was added to each sample. Samples were incubated at 68°C for 4h while shaking (1300rpm) to reverse crosslinks and digest proteins. DNA was purified on column using the QIAquick® PCR purification kit (Qiagen)

following the manufacturer’s protocol. DNA was eluted in 33µl of EB buffer. qRT-PCR was performed using the Light Cycler 480 System (Roche). Data were analysed using the Light Cycler 480 Software Release 1.5.0 SP1 (Roche).